source: https://doi.org/10.7892/boris.117848 | downloaded: 1.2.2022
DOI 10.1007/s00424-003-1129-5
I O N C H A N N E L S , T R A N S P O R T E R S
Rieko Sakai · Claudia Elfgang · Rolf Vogel · Klaus Willecke · Robert Weingart
The electrical behaviour of rat connexin46 gap junction channels expressed in transfected HeLa cells
Received: 7 May 2003 / Accepted: 29 May 2003 / Published online: 12 July 2003 Springer-Verlag 2003
Abstract Pairs of human HeLa cells expressing rat connexin46 were used to study the electrical properties of gap junction channels with the dual voltage-clamp method. The steady-state conductance (gj,ss) had a bell- shaped dependence on transjunctional voltage (Vj). The parameters of the Boltzmann fit were: Vj,0=42 mV, gj,min=0.12, z=2.5 (pipette solution: K+ aspartate; 27 C).
The Boltzmann parameters were sensitive to the ionic composition of the pipette solution (KCl, K+ aspartate, TEA+Cl, TEA+ aspartate). The Vj-dependent inactiva- tion of the junctional current Ij was approximated by single exponentials (exceptions: two exponentials with KCl atVj75 mV and K+aspartateatVj=125 mV). The time constant of inactivation (ti) decreased with increas- ingVjand was sensitive to the pipette solution. The larger the ions, the slower the inactivation. Recovery from inactivation followed a single exponential. The time constant of recovery (tr) increased with increasing Vj. Single-channel currents showed a main state, several substates and a residual state. The corresponding con- ductances gj,main and gj,residual decreased slightly with increasingVj; extrapolation toVj=0 mV yielded values of 152 and 28 pS, respectively (K+ aspartate; 37 C). The values of gj,main and gj,residual were dependent on pipette
solution. The ratiogj,main/gj,residual increased with increas- ing ionic size, suggesting that the residual state impairs ion permeation more severely than the main state. The gj,main data suggest that the ionic selectivity of Cx46 channels may be controlled primarily by ionic size.
Compared with hemichannel results, docking of connex- ons may modify the channel structure and thereby affect the ionic selectivity of gap junction channels. The open channel probability at steady state (Po) decreased with increasingVj. The parameters of the Boltzmann fit were:
Vj,0=41 mV,z=2.2 (K+aspartate; 27 C).
Keywords Gap junction · Connexin46 · Electrical properties · Conductance · Kinetics · Lens
Introduction
The lens of the vertebrate eye consists of a core of regularly packed fibre cells and a layer of epithelial cells covering the anterior surface of the organ. A unique feature of this organ is the absence of a vascular system.
To maintain tissue homeostasis, the lens relies on metabolic co-operation via gap junctions. Extensive communication via gap junctions exists between the epithelial cells, between the fibre cells and between the two cell types. This has been documented by morpho- logical and functional studies (see [12]). Immunolocal- ization and molecular biology techniques have identified three gap junction proteins in the lens, the connexins Cx43, Cx46 and Cx50. While epithelial cells express Cx43 [2, 20], fibre cells produce Cx46 and Cx50 [21, 36].
Functional aspects of Cx46 and Cx50 gap junctions have already been examined in pairs ofXenopus oocytes injected with rodent mRNA and information obtained on the voltage sensitivity of the gap junction conductance [11, 36, 37]. However, large membrane current noise has prevented the characterization of single channels. To circumvent this problem, attempts have been made to isolate cell pairs from rodent lenses using enzymatic treatments. While this approach has been successful for R. Sakai · R. Weingart (
)
)Department of Physiology, University of Bern, Bhlplatz 5, 3012 Bern, Switzerland
e-mail: weingart@pyl.unibe.ch Tel.: +41-31-6318706 Fax: +41-31-6314611 R. Sakai
Brain Science Institute,
The Institute of Physical and Chemical Research (RIKEN), Wako-shi, 351-0198 Saitama, Japan
C. Elfgang · K. Willecke
Institute of Genetics, University of Bonn, 53117 Bonn, Germany
R. Vogel
Cardiology, Swiss Cardiovascular Centre Bern, University Hospital, Inselspital, 3008 Bern, Switzerland
epithelial cells [8], difficulties have been experienced with fibre cells [9]: co-expression of two connexins renders it difficult to determine the electrical properties of identified gap junction channels.
The aim of this study was to explore the electrical properties of Cx46 gap junction channels in HeLa cells transfected with cDNA coding for rat Cx46. The exper- iments were carried out on cell pairs with variable intercellular coupling. Multi-channel and single-channel currents were recorded under different ionic conditions and the conductive and kinetic properties determined.
Preliminary data have been published elsewhere in abstract form [22].
Materials and methods
Cells and culture conditions
Experiments were performed with a clone of human HeLa cells transfected with a cDNA construct containing the coding sequence of rat Cx46. The 1592-bp EcoRI fragment of rat Cx46 cDNA [21]
was ligated into the expression vector pBEHpac18 [15] that contained the SV40 early promoter, a polyadenylation signal and a gene conferring resistance to puromycin. In the Cx46 cDNA fragment all ATG codons upstream of the start codon had been removed. HeLa cells were transfected with 20 g recombinant connexin46-pBEHpac18 plasmid using the calcium phosphate transfection protocol [6]. Between 24 and 48 h after exposure to DNA, the antibiotic puromycin (1 g/ml) was added to the medium.
Clones were selected after 3 weeks, grown in DMEM (Gibco, Paisley, UK) containing 10% FCS and 1 g/ml puromycin (Sigma, St. Louis, MO, USA), passaged weekly and diluted 1:10. For experiments, monolayers of cells (~4105cells/cm2) were harvest- ed and re-suspended in DMEM containing 10% FCS (0.2–
1106cells/ml). Thereafter, the cells were seeded onto sterile glass cover-slips (~104cells/cm2) placed in multi-well culture dishes and used within 24 h after plating.
Solutions
Experiments were carried out in modified Krebs-Ringer solution (mM): NaCl 150, KCl 4, CaCl2,2, MgCl21, glucose 5, pyruvate 2, HEPES 5 (pH 7.4). Patch pipettes were filled with normal pipette solution (in mM): K+aspartate120, NaCl 10, MgCl21, CaCl21, HEPES 5 (pH 7.2), EGTA 10 (pCaffi8), MgATP 3, through a 0.22- m-pore filter. In some experiments, K+aspartatewas replaced by equimolar amounts of KCl, TEA+ Cl or TEA+ aspartate. The specific conductance of the pipette solutions was determined with a conductivity meter (CDM 83; Radiometer, Copenhagen, Denmark) at 27 C.
Electrical measurements
Cover-slips with adherent cells were transferred to a chamber superfused at 2 ml/min with saline at 27 C. To alter the temperature, saline reservoirs were kept in heated or cooled vessels. The temperature in the chamber was measured with a thermistor positioned close to the cells. The chamber was mounted on the stage of an inverted microscope equipped with phase- contrast optics (Diaphot-TMD, Nikon; Nippon Kogaku, Tokyo, Japan). Patch pipettes were pulled from glass capillaries (GC150F- 10; Harvard Apparatus, Edenbridge, UK) using a horizontal puller (DMZ-Universal; Zeitz Instruments, Munich, Germany). When filled with solution, they had DC resistances of 3–6 MW. Exper- iments were carried out on cell pairs formed in culture [28]. Each
cell of a pair was attached to a pipette. After establishing a GW-seal, the membrane patch was disrupted enabling whole-cell recording.
The pipettes were connected to separate amplifiers (EPC7; List Electronic, Darmstadt, Germany). A dual voltage-clamp method allowed the membrane potential of each cell to be controlled (V1, V2) and the current flow through each pipette to be measured (I1, I2). Initially, the membrane potential of both cells was clamped to 30 mV. Thereafter, a junctional voltage (Vj) was established by de- or hyperpolarizing cell 1 while maintaining the membrane potential of cell 2. Under these conditions, Vjcorresponds to the voltage between the cells,Vj=V2V1. The current recorded from cell 1 represents the sum of two components, a gap junction current (Ij) and a non-junctional membrane current (Im,1), the current recorded from cell 2 is thus Ij. Voltage and current signals were stored on FM tape (SE 3000; SE Lab, Feltham, UK). For analysis the signals were filtered at 1 kHz (8-pole Bessel filter) and digitized at 2 kHz (12-bit A/D converter IDA 12120; INDEC Systems, Capitola, Calif., USA). Data acquisition and analysis were performed with the software C-Lab (INDEC Systems). Curve fitting and statistical analysis were done with SigmaPlot and SigmaStat, respectively (Jandel Scientific, Erkrath, Germany). The results are presented as means€SEM. The significance of differences between means was established using Student’st-test.
Biophysical modelling
Electrical properties of gap junction channels were modelled by two different approaches. The first method relies on macroscopic data and uses a top-down algorithm by assuming hemichannel properties (VW-model) [33]. The second method uses a bottom-up algorithm such as the Poisson-Nernst-Planck formulation of electrodiffusion, which is based on the combined treatment of selectivity and permeation (PNP-model) [7]. This model has been applied previously to describe the properties of single ion channels [7] as well as single gap junction channels and hemichannels [24].
It considers local chemical interactions depicted by an offset in chemical potential, which probably reflects differences in dehy- dration, solvation and rehydration energies associated with entry and exit steps of channel permeation. These interactions are modelled by fixed charges distributed along the channel. The purpose of the latter simulation is to demonstrate that specific charge profiles are able to reproduce the essential features of Cx46 single-channel conductance. However, it would have been beyond the scope of the present work to derive a set of parameters representing the best fit to the problem.I/V-curves were calculated using the code solving the PNP equation available at http://
www.pnponline.org./program.php. The PNP solver is accessed via a graphical user interface. The settings of the PNP parameters were as follows: pore length=100 , left ion concentration=120 mM, right ion concentration=120 mM, diffusion coefficient of cat- ion=2.048105 cm2/s (K+), diffusion coefficient of an- ion=0.655105 cm2/s (aspartate), ambient temperature=27 C, relative dielectric constant of aqueous pore=80. For the comparison with biological data, theI/V-curves were transformed into conduc- tance/voltage curves.
Results
Voltage dependence of gap junction currents
Voltage pulses of long duration (10 s), variable amplitude (150 mV) and either polarity were applied to one cell of the pair while the junctional currentIjwas recorded from the other. Figure 1A shows records obtained with the pipette solution containing K+aspartateas major charge carriers. Hyperpolarization of cell 2 by 75 mV (V2, left- hand side) provoked an Ij with time-dependent inactiva-
tion (I1, left-hand side). Depolarization of the same magnitude led to a similarIj of opposite polarity (V2and I1, right-hand side). To perform a complete experiment, the transjunctional voltage Vj was altered stepwise between €10 and €150 mV. Figure 1B summarizes the results from ten cell pairs. The amplitude of Ij was determined at the beginning (Ij,inst; inst: instantaneous) and end (Ij,ss; ss: steady state) of eachVjpulse to calculate the conductances gj,inst=Ij,inst/Vj and gj,ss=Ij,ss/Vj, respec- tively. On the one hand, the values of gj,inst were normalized with respect to the maximalgj,instat smallVj
gradients. The normalized values ofgj,instwere plotted as a function of Vj (gj,inst=f(Vj), open circles). On the other hand, the values of gj,ss were normalized with respect to the gj,instprevailing at eachVj. The normalizedgj,sswere plotted as a function of Vj (gj,ss=f(Vj), solid circles). Cell pairs with gj,inst>2 nS were excluded from the analysis to minimize series resistance problems [3, 35].
The gj,inst data exhibited a slight curvature with a symmetry at Vj=0 mV. The smooth curve represents the best fit of data to:
gj;inst¼ Gj
e
Vj
VH 1þe Vj VH
! þe
Vj
VH 1þe Vj
VH
! ð1Þ
(VW-model; Eq. 29 in [33]). Gj is a dimensionless fitting parameter and VH a decay constant. The analysis yielded the following values: Gj=2.0; VH=159 mV. At Vj=0 mVgj,instwas 1.0 and 0.85 at Vj=€100 mV.
The gj,ss data followed a bell-shaped relationship which was nearly symmetrical. At Vj=0 mV, gj,ss was maximal. Between Vj=€15 and €75 mV, it decreased steeply. Beyond €100 mV, it remained virtually constant.
The smooth curve represents the best fit of data to the Boltzmann equation applied separately to each voltage polarity:
gj;ss¼ 1gj;min
1þe A VjVj;0
þgj;min ð2Þ
wheregj,minis the normalized conductance at largeVjand Vj,0 corresponds to the Vj at which gj,ss/gj,inst is half- maximally inactivated. Ais a constant expressing gating charge, zq(kT)1 [13]. The values obtained from the analysis are given in Table 1 (pipette solution: K+ aspartate).
The voltage dependence ofIjwas also examined in the presence of pipette solutions containing KCl, TEA+Clor TEA+ aspartate as major charge carriers (signals not shown). The records obtained indicated distinct differ- ences inVjsensitivity ofIj. Figure 2 shows the results of the analysis plotting the normalized gj,ss vs.f(Vj) for the three solutions: KCl (circles, solid curve); TEA+ Cl(squares, dashed curve); TEA+ aspartate (triangles;
dotted curve). The smooth curves represent the best fit of data to the Boltzmann equation. The values obtained from the analysis are given in Table 1. A comparison of the data indicates subtle differences caused by the ions of the pipette solutions.
Table 1 Electrical properties of multi-channel gap junction cur- rents. Means€SEM (in parentheses) for negative/positive transjunc- tional potentialVj(gj,minis the normalized conductance at largeVj
Pipette solutions Vj,0(mV) gj,min z n
KCl 32.4/30.4 0.15/0.15 2.6/2.7 10
(1.1/1.6) (0.01/0.02) (0.2/0.3) K+aspartate 42.1/41.6 0.12/0.12 2.5/2.5 10
(0.5/0.7) 0.01/0.01 (0.1/0.1)
TEA+Cl 45.7/44.0 0.10/0.10 2.2/2.5 9
(0.6/0.5) (0.01/0.01) (0.1/0.1) TEA+aspartate 45.1/45.2 0.07/0.07 2.4/2.3 7
(0.5/0.6) (0.01/0.01) (0.1/0.1) The following differences were not significant statistically (Stu- dent’st-test):Vj,0data: TEA+Clvs. TEA+aspartate;zdata: KCl vs. K+aspartate, K+aspartatevs. TEA+Cl, TEA+Clvs. TEA+ aspartate
Fig. 1A, B Dependence of the multi-channel currents Ij on transjunctional voltage Vj in pairs of HeLa cells expressing rat connexin Cx46 gap junctions. A Responses of Ij to Vj. V1, V2: membrane potential of cell 1 and cell 2;I1: current measured from cell 1.DeflectionsinV2andI1correspond toVjandIj, respectively.
Vj=€75 mV. B Normalized gap junction conductance, gj(norm), determined at the beginning (gj,inst; ) and end (gj,ss; l) of Vj
pulses, as a function of Vj(ten cell pairs). Thecurves gj,inst=f(Vj) andgj,ss=f(Vj) show the best fits to Eqs. 1 and 2, respectively. For the Boltzmann parameters, see Table 1
Inactivation of gap junction currents
Next we explored the kinetics of Ij inactivation using pipette solution with K+ aspartate. Figure 3 shows signals gained at Vj=75 mV (left-hand panel) and Vj=125 mV (right-hand panel). The velocity of Ij inactivation was sensitive to the amplitude of Vj, with acceleration asVjincreased. To analyse the time course of inactivation, Ij signals were subjected to least-squares curve fitting. At Vj=75 mV, the best fit was achieved with a single exponential:
IjðtÞ ¼Ij1e
ti1t þIj;ss ð3Þ
where Ij(0) corresponds to Ij at time t=0 and equals Ij1+Ij,ss; ti1 is the time constant of Ij inactivation. At Vj=125 mV, the sum of two exponentials provided a better fit:
IjðtÞ ¼Ij1e
ti1t þIj2e
tti2 þIj;ss ð4Þ
In this case, Ij(0) equals Ij1+Ij2+Ij,ss. The smooth curves indicate the best fit ofIjto Eqs. 3 and 4, respectively. The analysis yielded the following values: ti1=240 ms (Vj=75 mV); ti1=83 ms, ti2=438 ms (Vj=125 mV). In the latter case,ti1andti2were different by a factor of 5.3, suggesting that this phenomenon is genuine.
Figure 4A summarizes the inactivation data from ten cell pairs. The values oftiwere obtained from individual Ijrecords, averaged and plotted as a function ofVj. ForVj
up to €100 mV, the analyses yielded single time constants, ti1, for larger values of Vj, fitting with two time constants was more appropriate, ti1 and ti2. The symbols correspond to mean values ofti1(ti2omitted for clarity). The values atVjof 25 mV were rejected due to the small Ij amplitude and the poor signal/noise ratio.
Over the voltage range analysed, ti1 decreased with increasingVj for both polarities.
Considering a two-state process (main state!residual state; see Single-channel conductances), the rate constants for channel opening (a) and closing (b) can be estimated according to [13]:
a¼
gj;ssðVjÞ
gj;instðVjÞgj;min 1gj;min
ti ð5Þ
and Fig. 3 Influence ofVjon the time course ofIjinactivation. Current traces I1obtained at Vj=75 mV (left) and 125 mV (right). The smooth curvesare the best fits of currents to Eqs. 3 and 4
Fig. 4A, B Kinetic properties of Ij inactivation. A Plot of time constants ofIjinactivation,ti1, as functions ofVj. Data from ten cell pairs. The curves were calculated from Eq. 9 taking into account the values for aand b determined by Eqs. 7 and 8.B The rate constants of channel opening, a (l), and closing, b () as functions ofVj. Thecurvesshow the best fits to Eqs. 7 and 8. For the kinetic parameters obtained, see Table 2
Fig. 2 Effects of intracellular ions on the dependence ofIjon Vj. The cells were dialysed with pipette solutions containing KCl (l), TEA+Cl(n) or TEA+aspartate(s) as major charge carriers. The normalized gap junction conductance at steady state,gj,ss, is plotted vs.Vj;n=10, 9 and 7 cell pairs, respectively. Thecurvesshow the best fits to Eq. 2.Solid curve: KCl;dashed curve: TEA+Cl;dotted curve: TEA+aspartate. For the Boltzmann parameters, see Table 1
b¼
1ggj;ssðVjÞ
j;instðVjÞ
1gj;min
ti
ð6Þ Using these equations, values for a and b were calculated from the data in Fig. 4A and plotted as functions of Vj (Fig. 4B). The graphs a=f(Vj) (solid circles) and b=f(Vj) (open circles) indicate that ti is governed byaat lowVjand bybat largeVj. The smooth curves represent the best fit of data to:
a¼le Aa VjVj;0
ð7Þ and
b¼le Ab VjVj;0
ð8Þ where Aa and Ab express the voltage sensitivity of the rate constants andlis a constant corresponding to the rate at which a=b (prevails at Vj=Vj,0) [13]. The analysis yielded similar values for negative/positiveVj:l=0.0013/
0.0014 s1; Aa=0.058/0.042 mV1; Ab=0.024/
0.022 mV1; Vj,0=40.2/36.5 mV. It follows that b>a when Vj>Vj,0 andb<a whenVj<Vj,0. The combination of Eqs. 7 and 8 enablesti1to be calculated:
ti1¼ 1
aþb ð9Þ
This relationship was used to plot the functionti1=f(Vj) for negative and positiveVj(continuous lines in Fig. 4A).
The resulting curves show a maximum at Vjffi50 mV.
There is a reasonable correlation between the curves and
the experimentally determined values of ti1 over the voltage range examined, i.e. Vj=€50 to €125 mV.
The kinetics ofIjinactivation were also studied in the presence of other pipette solutions. Figure 5 shows current records obtained with KCl (top), TEA+Cl(middle) and TEA+ aspartate(bottom).Vjpulses of 50 mV amplitude and 10 s duration were used (V1=30 mV,V2=80 mV).
In the case of KCl, TEA+ Cl and TEA+ aspartate, inactivation followed a single exponential with a ti1 of 333, 417 and 500 ms, respectively (smooth curves).
Hence, inactivation of Ij was affected by the ions of the pipette solution. Figure 6 summarizes the data obtained with these solutions (KCl: n=10; TEA+ Cl: n=9; TEA+ aspartate: n=7). The time constants extracted were sampled, averaged and plotted on a logarithmic scale as a function of Vj (Fig. 6A). Negative and positive Vj
yielded similar ti values and hence were pooled. In the case of TEA+ Cl (squares) and TEA+ aspartate (trian- gles), a single time constant was obtained at each Vj. In the case of KCl (circles), a single time constant was found atVj=50 mV and two atVj75 mV (for clarity, onlyti1is shown). The different pipette solutions gave rise to Fig. 5 Influence of pipette solution on the time course of Ij
inactivation. Ij records were obtained with pipette solutions containing KCl (top), TEA+ Cl (middle) and TEA+ aspartate (bottom). Vj=50 mV (V1=80 mV, V2=30 mV), pulse dura- tion=10 s. The analyses yielded single time constants. Thesmooth curvesare the best fits of currents to Eq. 3
Fig. 6A, B Effects of intracellular ions on kinetic properties ofIj
inactivation.ATime constants ofIjinactivation,ti, as functions of Vj. Values ofti1obtained at negative and positiveVjwere pooled and averaged (lKCl,n=10;nTEA+Cln=9;sTEA+aspartate n=7). Thesmooth curveswere calculated from Eq. 9. Thedashed curveshows the K+aspartatedata from Fig. 4A after pooling.B Rate constants of channel opening,a(solid symbols) and channel closure,b(open symbols), deduced from the time constants inA, as functions ofVj. Thesmooth curvesshow the best fits to Eqs. 7 and 8. Thedashed curveshows the K+aspartatedata from Fig. 4B after pooling. For the kinetic parameters obtained, see Table 2
distinct relationships betweenti1andVj. In each case,ti1 decreased with increasing Vj.
Equations 5 and 6 were then used to calculate the rate constants a and b for the different pipette solutions.
Figure 6B shows the resulting plots. Solid and open symbols correspond to a and b, respectively. The solid curves represent the best fit of data to Eqs. 7 and 8. The fitting parameters obtained from the analysis are summa- rized in Table 2. For comparison, Fig. 6B and Table 2 also include the kinetic data for K+aspartatesolution already shown in Fig. 4B (dashed curves, symbols omitted). This involved pooling of the values for positive and negative Vj. According to Fig. 6B,b=f(Vj) increased with increas- ing Vj while a=f(Vj) decreased. The increase in b was dependent on the ionic composition of the pipette solution. The smaller the ions, the more pronounced the effect. In contrast, the decrease in a was virtually insensitive to the ions. Hence, the increase inti1brought about by the substitution of KCl for TEA+aspartatecan be explained with an increase inb.
The rate constants gained for KCl, TEA+ Cl and TEA+ aspartate were then used to calculate ti1 as a function of Vj using Eq. 9. The continuous curves in Fig. 6A illustrate the results. The functionti1=f(Vj) for K+ aspartate, already shown in Fig. 4A, are also included (dashed curve, symbols omitted). For this purpose, the data for negative and positive Vjwere pooled. The graph shows a distinct correlation betweenti1and the ions of the pipette solution for voltages larger than 50 mV. The solutions containing the smallest ions, i.e. KCl, gave rise to the smallest ti1 values, the solution with the largest ions, i.e. K+aspartate, gave rise to the largestti1values.
Recovery ofIj from inactivation
We have also examined the recovery of Ij from inacti- vation using pipette solution with K+ aspartate. The upper panel in Fig. 7A shows the pulse protocol. A conditioning pulse applied to cell 1 (pulse 1) was followed by a test pulse (pulse 2) after a variable delay.
Both pulses produced a Vj of 100 mV amplitude and 5 s duration. They allowed Ij to inactivate maximally and reach a steady state. The lower panel shows superimposed traces indicating that Ij,inst of pulse 2 grew larger with increasing inter-pulse interval. To examine the role ofVj
onIjrecovery, we also used pulses of different amplitude (traces not shown). Figure 7B summarizes the results obtained. For each pulse, the current inactivated was calculated, i.e. Ij,instIj,ss. Thereafter, the values of Ij,instIj,ssassociated with pulse 2 were normalized relative to Ij,instIj,ss associated with pulse 1 and plotted as a function of the inter-pulse interval. The data illustrated were obtained at Vj=50 mV (squares; n=5), 75 mV (triangles;n=11) and 100 mV (circles;n=8). The smooth curves correspond to the best fit of data to a single exponential:
ðIj;instIj;ssÞpulse2
ðIj;instIj;ssÞpulse1¼1eð Þttr
ð10Þ where tris the time constant of recovery andt the inter- pulse interval. The analysis yielded the following values for tr: Vj=100 mV: 662 ms; Vj=75 mV: 404 ms;
Vj=50 mV: 111 ms. Hence, the larger the inactivation of Ij, the slower the recovery.
Table 2 Inactivation parameters of multichannel gap junction currents. Values ofl,Vj,0,Aaand Abwere deduced from the data in Fig. 6B (lconstant corresponding to the rate at whicha=b, i.e. at Vj=Vj,0, a, b rate constants of channel opening and closure, respectively, Aa, Ab voltage sensitivity of the respective rate constants)
Pipette solutions l(s1) Vj,0(mV) Aa(mV1) Ab(mV1)
KCl 0.0014 26.5 0.056 0.026
K+aspartate 0.0014 39.3 0.062 0.023
TEA+Cl 0.0015 41.2 0.065 0.019
TEA+aspartate 0.0013 39.0 0.058 0.014
Fig. 7A, B Recovery of Ij from inactivation. A Double pulse protocol used to determineIjassociated with a test pulse (pulse 2) applied at different times after a conditioning pulse (pulse 1).V1, V2: superimposed membrane potentials of cells 1 and 2;I2: family of current traces from cell 2.DeflectionsinV1andI2correspond to Vj and Ij, respectively. B (Ij,instIj,ss)pulse2/(Ij,instIj,ss)pulse1 as a function of inter-pulse interval. Data were obtained atVj=50 mV (n; 5 cell pairs), 75 mV (s; 11 cell pairs) and 100 mV (l; 8 cell pairs). Thesmooth curvesare the best fits to Eq. 10
Single-channel conductances
Single-channel events were studied in cell pairs with gap junctions consisting of between one and three operational channels. The protocol involved repetitive application of Vj pulses of 200–500 ms duration, different amplitude (100 mV) and either polarity. Figure 8A shows signals using pipette solution with K+aspartate. Hyperpolariza- tion of cell 1 (V1) led to aVj of 25, 50 and 75 mV. The associatedIjsignals (I2traces from top to bottom) showed discrete levels corresponding to the main state, the residual state (dotted lines) and short-lived substates in between. During Vj pulses, Ij did not return to the reference level (solid lines) indicating that the channels failed to close completely. The analysis yielded the following conductances (gj,main/gj,residual): 136/23, 130/20 and 118/18 pS, forVjof 25, 50 and 75 mV, respectively.
The histograms in Fig. 8B summarize the results from 14 cell pairs plotting the number of events vs. conduc- tance. The values ofgj,residualandgj,mainwere pooled in 2- and 4-pS bins, respectively. To avoid interference from the sensitivity to Vj (see below), this analysis included data at Vj=€50 mV only. The left- and right-hand distributions correspond to gj,residual and gj,main, respec- tively. Both data sets showed a binomial distribution approximated by a Gaussian function (smooth curves).
The mean values ofgj,mainandgj,residualwere 127.5€0.4 pS (n=471) and 19.0€0.3 pS (n=175), respectively.
To assess the relationships betweenVjandgj,Ijrecords gained at different Vj were considered. Values of gj,main
and gj,residual were sampled, averaged and plotted as a function of Vj. Figure 9B illustrates the relationships gj,main=f(Vj) (open circles) and gj,residual=f(Vj) (solid cir- cles).gj,mainshowed a slight dependence onVj, i.e. it was maximal atVj=0 mV and decreased as Vj was increased.
gj,main was fitted to the function:
gj;main¼ GH
e
Vj VH 1þe
Vj VH
! þe
Vj
VH 1þe Vj
VH
! ð11Þ
(VW-model; Eq. 29 in [33]).GHis the conductance of a hemichannel in the high-conductance state, i.e. the main state, at Vj=0 mV. The analysis yielded the following values:GH=276 pS;VH=129 mV.gj,residualalso showed a moderate dependence on Vj, i.e. it decreased with increasing Vj. Reliable measurements were obtained for Vjbetween €25 and €100 mV. At Vj<€25 mV, gj,residual events were rare and too small to be resolved (see below).
gj,residualdata were fitted using a standard error minimiza- tion procedure. The analysis gave the following values:
GL=25 pS,VL=244 mV. GLrepresents the conductance of a hemichannel in the low-conductance, i.e. residual, state, at Vj=0 mV [33].
Fig. 8A, B Single-channel currents.AResponses ofIjtoVj=25, 50 and 75 mV (I2signals from totop tobottom).V1,V2: membrane potential of cells 1 and 2; I2: current measured from cell 2.
Deflections in V1 and I2 correspond to Vj and Ij, respectively.
Continuous lines indicate zero current, dotted lines the residual current. The analysis yielded the following conductances (gj,main/ gj,residual): 136/23 pS; 130/20 pS; 118/18 pS, respectively. B Histogram of single-channel conductances. The analysis included values obtained atVj=€50 mV (14 cell pairs). The left and right hand distributions reflectgj,residualandgj,maindata, respectively. The smooth curves show the best fits to Gaussian functions. For the values obtained, see Table 3
Fig. 9A, B Relationship between single-channel conductances,gj, andVj. Comparison of simulations by the VW- and PNP-models.A Charge density profile of the PNP-model. The graph shows two negative charge regions (2 mol/l) located at the pore endings, each occupying 10% of the pore length.BConductancesgj,main() and gj,residual(l) as functions ofVj(14 cell pairs). Means€SEM,n=471 and 175 determinations, respectively. Mosterror barsare smaller than the symbols. Thesolid curvescorrespond to the relationships gj,main=f(Vj) and gj,residual=f(Vj) and show the best fits to the VW- model. Thedashed curve shows the gj,maindata simulated by the PNP-model, assuming a pore radius of 5.3
Since a gap junction channel consists of two hemichannels in series, gj,main and gj,residual at Vj=0 mV can be calculated as GH/2=138 pS and (GHGL)/
(GH+GL)=23 pS, respectively. Taking into account the values of Q10 (see Influence of temperature on gap junction currents), gj,main andgj,residualat 37 C would be 152 and 28 pS, respectively.
To explore the ionic permeability of Cx46 channels, unitary currents were also recorded with pipette solutions containing KCl, TEA+ Cl or TEA+ aspartate. In the presence of TEA+ aspartate, Ij records often showed critical signal/noise ratios. In these cases, the currents were analysed using an all-point procedure. Figure 10A shows selected Ij traces elicited by a standard Vj pulse (50 mV, 400 ms). Each trace exhibited two prominent levels attributable to the main and residual states (dotted lines). In the presence of KCl, gj,main and gj,residual were 207 and 33 pS, respectively (top). With TEA+ Cl, the respective conductances were 108 and 13 pS (middle), with TEA+aspartate, 65 and 6 pS (bottom). Hence,gj,main and gj,residual decreased with increasing ionic size of the major charge carriers. Figure 10B summarizes the data.
Values ofgj,residualandgj,mainwere sampled in 2- and 4-pS bins, respectively, and plotted as frequency histograms.
The two distributions correspond to thegj,residualandgj,main data. The smooth curves represent the best fit of data to a Gaussian function. Table 3 summarizes the results of the
analysis and includes the values for K+ aspartate (see Fig. 8B).
Figure 11 shows a normalized plot of the channel conductance,gj, as a function of the conductance of the pipette solution,s. The values of gjwere taken from the experiments described above, the values of s were measured with a conductivity meter (see Table 3). The KCl pipette solution served as reference. The symbols correspond togj,main(open circles) andgj,residualdata (solid circles). The dotted line represents the function s=m·gj
with m=1, assuming identical ionic mobility in the channel pore and in bulk solution. Neither gj,main nor gj,residual fulfilled this prediction, suggesting interactions between the ions and the channel structure.
Simulations with the PNP-model
For calculations with the PNP-model, the gap junction channel is divided into ten segments of piecewise constant and fixed charges. Segments 1 and 10 include the intracellular endings of the channel, while segments 5 Fig. 10A, B Influence of intracellular ions on single-channel
currents. The cells were dialysed with pipette solutions containing KCl (top), TEA+ Cl (middle) and TEA+ aspartate (bottom) as major charge carriers.AResponses ofIjto aVjpulse of 50 mV and 400 ms (V1=80 mV,V2=30 mV).Continuous linesindicate zero current, dotted lines residual current. B Histograms of single- channel conductances. The analysis included values obtained at Vj=€50 mV only. The left- and right-hand distributions reflect gj,residualandgj,maindata, respectively. Thesmooth curvesshow the best fits to Gaussian functions. For the values obtained, see Table 3
Table 3 Single-channel conductances (gj) for the main and residual conductance states. Means€SEM. Valuesinparentheses give the number of cell pairs and the number of observations. Channel conductances were obtained atVj=50 mV (sconductance of pipette solution)
Pipette solutions gj,main(pS) gj,residual(pS) s(mS/cm)
KCl 212.7€0.9 31.5€0.4 17.5
(8/248) (8/71)
K+aspartate 127.5€0.4 19.0€0.2 13.5 (14/471) (14/175)
TEA+Cl 108.8€0.4 12.3€0.1 13.0
(5/209) (5/71)
TEA+aspartate 65.7€0.6 6.1€0.4 8.4 (10/307) (10/75)
Fig. 11 Selective permeability of single channels. Plot of the normalized channel conductancesgj,main() andgj,residual(l) vs.
normalized conductance of the pipette solution,s. Pipette solution containing KCl was used as reference. The dotted line indicates identical ionic mobility in the channel pore and in bulk solution
and 6 represent the docking area of the hemichannels.
Independent of charge polarity, gj,main increased with increasing |Vj| when charges were fixed near the docking zone, i.e. in segments 4–6 (data not shown). However, the opposite behaviour can be simulated by placing fixed charges of either polarity close to the proximity of the cytosolic channel ending, i.e. in segments 1, 2, 9 and 10.
As determined experimentally, gj,main of a Cx46 channel decreases from about 135 to 110 pS when Vjis increased from 25 to 100 mV (see Fig. 9B). This behaviour can be approximated by the PNP-model when fixed negative charges are placed in segments 1 and 10.
Positive charges that produced conductances of the same order are very small and thus the channel conductance becomes almost independent of Vj. The graph in Fig. 9A shows the charge density profile comprising two nega- tively charged regions (2 mol/l), each occupying 10% of the pore length and located towards the intracellular end.
Figure 9B includes the result of the respective simulation.
Choosing a pore radius of 5.3 (dashed curve) resulted in a satisfactory agreement between the experimental data and the PNP-model.
Open channel probability
At steady state the channels flickered primarily between the main state and residual state. Hence, single-channel records allowed the determination of the probability of a channel’s being in the main state, Po. The pulse protocol involved the application of aVjof long duration (20–30 s) and different amplitude. The initial segment of each Ij
record was discarded to avoid interference from time- dependent inactivation and substates [28]. Figure 12A shows records gained with the K+ aspartate pipette solution. The currents were elicited by a Vj of 25 mV (top), 50 mV (middle) and 100 mV (bottom). They indicate that the dwell time at discrete levels is correlated with the amplitude ofVj. At 25 mV, the channel flickered rarely and was preferentially in the main state. At 50 mV, it flickered more frequently and spent less time in the main state and more time in the residual state. At 100 mV, it flickered rarely and was mainly in the residual state.
The records in Fig. 12A and others were used to assess Po. The time in the main state was determined and divided by the record duration. The values of Po were averaged and plotted vs. Vj (Fig. 12B). Po decreased sigmoidally from 1 to 0 as Vj increased from 0 to 100 mV. The continuous curve represents the best fit of data to the Boltzmann equation using the following values:
Vj,0=40.6€1.9 mV;z=2.2€0.3 (ten cell pairs; see Voltage dependence of gap junction currents). ThisVj,0is close to that obtained from the function gj,ss=f(Vj), i.e. 41.4 mV (see Table 1).
Influence of temperature on gap junction currents To obtain further insight into the biophysical properties of Cx46 channels, the effect of temperature on Ij was examined with pipette solution containing K+ aspartate. Figure 13A shows segments of continuous records gained at different temperatures. To determine Ij,inst andIj,ss, Vj pulses of 50 mV amplitude and 10 s duration were delivered to cell 1 and the currents displayed at expanded time scale. To monitor the transitions between tempera- tures,Vjpulses of 25 mV amplitude and 200 ms duration were applied and the signals presented on a compressed time scale.
At 17 C (left-hand side), Ij inactivated slightly with time (gj,inst=1.23 nS,gj,ss=0.75 nS,ti1=1,100 ms). At 37 C (middle), it inactivated substantially due to dispropor- tionate increases of Ij,inst and Ij,ss (gj,inst=1.77 nS, gj,ss=0.48 nS, ti1=256 ms). Return to 17 C (right hand side) led to a recovery (gj,inst=1.21 nS, gj,ss=0.69 nS, ti1=714 ms). In five cell pairs, Vj was altered systemat- ically to obtain a family of Ij records at 17 and 37 C.
Figure 13B summarizes the results. The ratios gj,ss/gj,inst
were averaged and plotted as functions ofVj. For clarity, only half of each data set is shown (positive Vj: 17 C data; negative Vj: 37 C data). The smooth curves represent the best fit of data to the Boltzmann equation Fig. 12A, B Effects ofVjon probability of the channel open state, Po. A Segments of Ij records from cell pairs with a single operational gap junction channel obtained at steady-state. Currents traces gained at Vj=25 mV (top), 50 mV (middle) and 100 mV (bottom). B Relationship between Poand Vj. Values of Powere determined from long-lasting records (20–30 s) during steady-state conditions. The continuous curve shows the best fit to the Boltzmann equation
for following values: 17 C: Vj,0=58.5 mV, gj,min=0.12, z=1.7; 37 C:Vj,0=38 mV,gj,min=0.15,z=3.3.
These parameters and those obtained at 27 C using the same pipette solution, i.e. K+aspartate(see Table 1), were used to assess the temperature coefficient, Q10, of the Boltzmann parameters. Averages of Vj,0, gj,min and z were plotted on a logarithmic scale vs. temperature (not shown). Over the range examined, i.e. 17–37 C, each parameter increased with increasing temperature. The data were analysed using:
FT ¼F17Q
T1710
½
10 ð12Þ
where Tis the temperature in C,Fthe parameter under investigation, and F17 the value of the parameter at the reference temperature, i.e. 17 C. The best fit of data was obtained for the followingQ10values: 1.25, 1.2 and 1.38 for Vj,0, gj,min andz, respectively.
Figure 14A shows unitary currents recorded at differ- ent temperature. An increase from 17 to 37 C (upper and lower I2 trace) led to an increase in conductance, i.e.
gj,mainincreased from 104 to 143 pS andgj,residualfrom 17 to 24 pS. This corresponds to a 1.38-fold and 1.41-fold increase, respectively. Figure 14B summarizes the results from 22 cell pairs including the data shown in Fig. 8B.
Individual values ofgj,main(n=1,739) andgj,residual(n=352) were averaged and plotted on a logarithmic scale vs.
temperature. Over the range examined, i.e. 17–37 C, gj,main (open circles) andgj,residual(solid circles) increased with increasing temperature. The solid lines represent the best fit of data to Eq. 12 using a Q10 of 1.1 and 1.2 for gj,main andgj,residual, respectively.
Discussion
Our data indicate that rat Cx46 channels resemble other vertebrate gap junction channels, e.g. Cx30 [28], Cx40 [4]
or Cx43 [27]. They exhibit several conductance states and are sensitive to Vj. We show that many of the basic electrical properties are dependent on the ionic compo- sition of the pipette solution. Control solution containing K+aspartatewas used to mimic physiological conditions.
During the interventions, these ions were replaced by TEA+ and/or Cl.
Voltage gating ofIj
Thegjof Cx46 gap junctions is gated byVj, but not byVm
(latter data not shown). With regard to Vj gating, the analysis furnished the relationships gj,inst=f(Vj) and gj,ss=f(Vj), which characterize the instantaneous and steady-state properties, respectively (see Fig. 1B). On the one hand,gj,instdecreased slightly with increasingVj, leading to a moderate curvature of gj,inst=f(Vj). A comparison of Figs. 1B and 9 (pipette solution: K+ aspartate) indicates that the relationships gj,inst=f(Vj) andgj,main=f(Vj) resemble each other, suggesting that the Fig. 14A, B Influence of temperature on single-channel currents.A
Responses ofIjto aVjpulse of 50 mV and 300 ms (V1=80 mV, V2=30 mV) obtained at 17 C (upper trace) and 37 C (lower trace). Thecontinuous linesindicate zero current, thedotted lines residual current. BChannel conductancesgj,main() and gj,residual
(l) on a logarithmic scale as functions of temperature. Thesloping linesshow the best fits to Eq. 12 using the temperature coefficients, Q10, of 1.1 and 1.2, respectively
Fig. 13A, B Influence of temperature on multi-channel currents.A Segments of continuous records gained at 17 C (left), 37 C (middle) and 17 C (right). To determine Ij,inst and Ij,ss, long Vj
pulses (10 s, 50 mV) were administered to cell 1. To monitor transitions between temperatures, shortVjpulses (200 ms, 25 mV) were applied. Note the differenttime scale.BPlot of normalized conductance at steady state,gj,ss, vs.Vj. Data were obtained at 17 C (l) and 37 C (). For clarity, only half of each data set is shown (five cell pairs each). Thesmooth curvesshow the best fits to Eq. 2.
For the Boltzmann parameters, see text
